Neoinitimal hyperplasia is a common complicating feature of atherosclerosis, restenosis after angioplasty, and transplant vasculopathy. At the cell level, over-growth of the arterial wall is triggered by inflammatory signals that promote progressive phenotypic changes resulting in enhanced cell proliferation and migration. Evidence suggests that this dedifferentiation process known as phenotypic modulation may occur in both medial smooth muscle cells and adventitial fibroblasts where the biochemical hallmark of cell reprogramming is alteration in expression of vascular smooth muscle (VSM) alpha-actin, a cytoskeletal protein important for regulation of vascular contraction and cell movement. Hence, elucidation of the mechanisms governing expression of the VSM alpha-actin gene in cells of myogenic and fibroblastic lineage may reveal molecular targets of clinical utility in the management of atherosclerotic plaque stability and/or restenosis after surgical intervention. The primary goal of this research is to define the gene regulatory function(s) of a novel group of single-stranded DNA and mRNA-binding factors known as Puralpha, Purbeta, and MSYI. These proteins have been implicated in coordinately suppressing VSM alpha-actin gene expression in myofibroblasts and smooth muscle cells by both transcriptional and post-transcriptional mechanisms. In this proposal, particular emphasis will be placed on the participation of Purbeta in these processes since this protein appears to be the key component required for repression of the VSM alpha-actin promoter in cultured cells and because its pattern of expression in vascular tissue specimens is consistent with a role in phenotypic modulation. In vitro and in vivo approaches will be used to test predictions arising from conceptual models of gene regulation by 1) mapping structural domains and chemical modifications required for interaction of Purbeta with relevant nucleic acid and protein-binding partners 2) analyzing the assembly and disassembly of Pur-containing regulatory complexes on the genomic VSM alpha-actin promoter, 3) evaluating the ability of Pur and Y-box proteins to inhibit translation of VSM alpha-actin mRNA, and 4) characterizing the time-course of expression of Puralpha, Purbeta, and MSY1 in mouse models of vascular disease and injury. This research will contribute to an improved understanding of a unique class of single-stranded nucleic acid-binding factors that may account for the remarkable plasticity of VSM alpha-actin expression and phenotypic modulation during vascular remodeling.